Stroke is a leading cause of death and disability in the United States, yet effective treatments for stroke remain limited. The studies proposed within intend to facilitate the search for therapeutic targets for stroke by elucidating the role of cerebrovascular sphingosine kinase (SphK) signaling in the mechanism by which hypoxic preconditioning (HPC) affords ischemic protection to the neurovascular unit following stroke. SphK is upregulated by hypoxia or ischemia, and studies in tissues such as the heart and kidney have linked SphK to ischemic protection through multiple signaling pathways. HPC protects against cerebral ischemia- induced neurovascular dysfunction, however, the mechanisms of this protection are not well characterized. Recently, we obtained exciting preliminary data indicating that in cerebral microvessels, HPC upregulates the SphK2 isoform, and this SphK activity regulates HPC-induced ischemic protection. Thus, the following specific aims are proposed: Aim 1: To elucidate the dependency of cerebrovascular ischemic tolerance on the sphingosine kinase isoforms. Using a SphK inhibitor and SphK2 knockout mice, we intend to demonstrate that cerebral HPC is mediated by increased expression of SphK2 and its activity within vascular endothelial cells. Furthermore, we will study the regulation of vascular permeability by SphK following ischemia and demonstrate that ischemic tolerance mediated by SphK is associated with the preservation ofthe blood-brain barrier. Additionally, we will examine the adherens junction proteins PECAM-1 and VE-cadherin to determine if SphK maintenance of the BBB is mediated through adherens junctions. Aim 2: To elucidate the relationship between SphK2, and HIF-1 and HIF-2 during the induction of an Ischemic tolerant state in cerebrovascular endothelial cells. Using lentiviral transduction of siRNA to the specific HIF isoforms in a microvascular endothelial culture, and HIF-2 knockout mice, we intend to show that vascular HIF mediates the induction of SphK signaling in response to HPC. We will also examine the possible role of SphK in the regulation of HIF expression following HPC in vitro and in vivo. The proposed experiments will provide an increased understanding of the signaling of SphK in cerebral ischemic protection and the pathways by which that protection is induced. Elucidating the mechanisms of neurovascular unit protection will facilitate the development of stroke therapeutics.